DNA immunization with plasmids expressing the insulin B chain protects RIP-LCMV and NOD mice from type 1 diabetes by induction of CD4+, IL-4+ regulatory lymphocytes. Our hypothesis is that targeting presentation of insulin-B to the MHC class II pathway will enhance protection and that the regulatory cells induced will be present in peripheral blood after immunization and can be used as a pre-clinical marker predicting vaccine success on an individual basis (aim 1). Furthermore, we hypothesize that the insB specific regulatory cells act in vivo by modulating antigen presenting cells in the pancreatic draining lymph node rendering them incapable of propagating the autoaggressive response (aim 2). Aim 1: Bringing DNA vaccines to treat T1D to the clinic. a. Use of limp II, DNA vaccine constructs that target presentation of antigen to the MHC class II pathway. Since we have demonstrated that induction of insB specific CD4 lymphocytes is essential and can transfer protection using our DNA vaccine strategy, this should increase safety (avoiding MHC class I restricted responses to insulin that are associated with enhanced T1D) and possibly efficacy. b. Predicting the success of vaccination on an individual basis. We intend to precisely define which vaccination induced CD4 response correlates with protection on a per-mouse basis and whether such a response can be reliably detected in peripheral blood. Aim 2: How do insB specific CD4 regulatory Iymphocvtes work? a. In vitro analysis. Effect of IL-4 producing CD4 regulatory lymphocytes on aggressive lymphocytes, beta-cells and APCs in vitro. We expect to map that regulation occurs via APCs based on our previous data. In vitro studies will corroborate this notion and evaluate other possibilities. b. In vivo analysis. How do regulatory cells suppress T1D in vivo? Trafficking and in situ in vivo studies, isolation of APCs from recipients of regulatory cells. c. In vitro induction of regulatory cells ad testing of in vivo function. Can insB specific regulatory cells be induced from naive lymphocytes in vitro? These studies should bring immunization with islet antigen closer to the clinic and should further our understanding of how regulatory CD4 cells act in vivo.